1. Field of the Invention
The present invention relates to polypeptides exhibiting follicle stimulating hormone activity, to methods for preparing such polypeptides, and to the use of such polypeptides in therapy, in particular in the treatment of reproductive disorders.
2. Description of Related Art
a. Gonadotropins
Follicle stimulating hormone (FSH) is a member of the family of gonadotrophins that play key roles in human fertility. The gonadotrophins, which also include luteinising hormone (LH) and chorionic gonadotrophin (CG), are heterodimers, each consisting of a common α-subunit (92 amino acids) and a unique β-subunit (111 amino acids in FSH). The amino acid sequences of the mature forms of the α- and β-subunits are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.
Human FSH has been isolated from pituitary glands and from postmenopausal urine (EP 322,438) and has been produced recombinantly in mammalian cells (U.S. Pat. No. 5,639,640, U.S. Pat. No. 5,156,957, U.S. Pat. No. 4,923,805, U.S. Pat. No. 4,840,896, U.S. Pat. No. 5,767,251, EP 211,894 and EP 521,586). The latter references also disclose the human FSH β-subunit gene. U.S. Pat. No. 5,405,945 discloses a modified human α-subunit gene comprising only one intron.
Liu et al., J Biol Chem 1993, 15; 268 (2): 21613-7, Grossmann et al., Mol Endocrinol 1996 10 (6): 769-79, Roth and Dias (Mol Cell Endocrinol 1995 1; 109 (2): 143-9, Valove et al., Endocrinology 1994; 135 (6): 2657-61, Yoo et al., J Biol Chem 1993 25; 268 (18): 13034-42), U.S. Pat. No. 5,508,261 and Chappel et al., 1998, Human Reproduction, 13 (3): 1835 disclose various structure-function relationship studies and identify amino acid residues involved in receptor binding and activation and in dimerization of FSH.
b. Use of Gonadotropins in Assisted Reproductive Techniques
The gonadotrophins play crucial roles in the reproductive cycle, and their use in exogenous therapies is essential for assisted reproductive techniques (ART), such as in vitro fertilization (IVF), IVF in conjunction with intracytoplasmic sperm injection (IVF/ICSI) and embryo transfer (ET), as well as for ovulation induction (OI) in anovulatory patients undergoing in vivo fertilization either naturally or through intrauterine insemination (IUI).
U.S. Pat. No. 4,589,402 and U.S. Pat. No. 4,845,077 disclose purified human FSH which is free of LH and the use thereof for in vitro fertilization. EP 322 438 discloses a protein with at least 6200 U/mg FSH activity which is substantially free of LH activity, and wherein the FSH α-subunit and the β-subunit, respectively, may be wild type or specified truncated forms thereof.
Prolonged therapy is necessary to achieve a therapeutic effect, typically for 8-10 consecutive days and sometimes up to 21 days to stimulate folliculogenesis in women, and for up to 18 months in hypogonadotrophic males to induce spermatogenesis. Recombinant hFSH is typically administered as an i.m. or s.c. daily injection, with consequent discomfort and potential for local injection site reaction. Decreasing the frequency of administration would facilitate therapy and render gonadotrophin administration more convenient, more tolerable and patient-friendly.
c. Glycosylation of FSH
The gonadotrophins are glycoproteins, with each sub-unit having asparagine-linked (N-linked) oligosaccharide side chains that are important for in vivo activity and function. Carbohydrate addition (glycosylation) to polypeptides is a post-translational event that results in the addition of sugar chains to specific asparagine (N-linked) or serine/threonine (O-linked) amino acids. In contrast to the invariant amino acid sequence of the protein portion of glycoproteins, the carbohydrate structures are variable, a feature referred to as microheterogeneity. For example, N-glycosylation sites on the same protein may contain different carbohydrate structures. Furthermore, even at the same glycosylation site on a given glycoprotein, different carbohydrate structures may be found. This heterogeneity is a consequence of the non-template-directed synthesis of carbohydrates.
N-glycosylation of proteins occurs specifically at the consensus pattern Asn-Xaa-Ser/Thr, and to a lesser extent at the consensus pattern Asn-Xaa-Cys, where Xaa can be any amino acid residue. However, the presence of a consensus tripeptide is not sufficient to insure that an asparagine residue will be glycosylated. For example, N-glycosylation of the Asn-Pro-Ser/Thr sequence occurs at a rate 50-times lower than the other consensus patterns of Asn-Xaa-Ser/Thr.
Human FSH contains four N-linked glycosylation sites: two on the common α-subunit at positions 52 and 78 and two on the β-subunit at positions 7 and 24. Carbohydrates attached to the α-subunit of FSH are critical for dimer assembly, integrity, secretion and signal transduction, whereas β-subunit carbohydrates are important for dimer assembly, secretion and clearance of the heterodimer from the circulation.
Galway et al., Endocrinology 1990; 127 (1): 93-100 demonstrate that FSH variants produced in a N-acetylglucosamine transferase-I CHO cell line or a CHO cell line defective in sialic acid transport are as active as FSH secreted by wild type cells or purified pituitary FSH in vitro, but lacked in vivo activity, presumably due to rapid clearance of the inadequately glycosylated variants in serum. D'Antonio et al., Human Reprod 1999; 14 (5): 1160-7 describe various FSH isoforms circulating in the blood stream. The isoforms have identical amino acid sequences, but differ in their extent of post-translational modification. It was found that the less acidic isoform group had a faster in vivo clearance as compared with the acidic isoform group, possibly due to differences in the sialic acid content between the isoforms. Moreover, Bishop et al. Endocrinology 1995; 136 (6): 2635-40 conclude that circulatory half-life appears to be the primary determinant of in vivo activity. These observations led to the hypothesis that the half-life of FSH could be increased by introducing additional glycosylation sites to increase the sialic acid content of the polypeptide.
d. FSH Variants
FSH agonists with increased half-lives have been developed by fusing the carboxyterminal peptide of hCG (CTP) to native recombinant human FSH (rhFSH). The CTP moiety consists of amino acids 112-118 to 145 with four O-linked glycosylation sites located at positions 121, 127, 132 and 138. U.S. Pat. No. 5,338,835 and U.S. Pat. No. 5,585,345 disclose a modified FSH β-subunit extended at the C-terminal Glu with the CTP moiety of hCG. The resulting modified analogue is stated to have the biological activity of native FSH, but a prolonged circulating half-life. U.S. Pat. No. 5,405,945 discloses that the carboxy terminal portion of the hCG β-subunit or a variant thereof has significant effects on the clearance of CG, FSH, and LH.
U.S. Pat. No. 5,883,073 discloses single-chain proteins comprised of two α-subunits with agonist or antagonist activity for CG, TSH, LH and FSH. U.S. Pat. No. 5,508,261 discloses heterodimeric polypeptides having binding affinity to LH and FSH receptors comprising a glycoprotein hormone α-subunit and a non-naturally occurring β-subunit polypeptide, wherein the β-subunit polypeptide is a chain of amino acids comprising four joined subsequences, each of which is selected from a list of specific sequences. Klein et al. (2003) discloses a single chain analogue of FSH with an increased half-life, wherein the α- and β-subunits are linked by an oligopeptide containing two N-linked glycosylation sites.
WO 01/58493 discloses 77 mutations that may be made in the α-subunit of FSH and 51 mutations that may be made in the β-subunit of FSH in an attempt to improve the in vivo half-life of FSH. WO 01/58493 discloses that the mutant α- and β-subunits may be used individually (1 additional glycosylation site) or in combination (2 additional glycosylation sites). The 128 candidate mutants were identified by using 50 models of the 3D structure of FSH that were generated based solely on the structure of hCG and a sequence alignment of FSH and hCG despite only 32% identity between the β-subunits of hCG and FSH. WO 01/58493 does not disclose the production or testing of any α- or β-subunits of FSH where a glycosylation site was introduced by site directed mutagenesis.
A clinical need exists for a product which provides part or all of the therapeutically relevant effects of FSH, and which may be administered at less frequent intervals as compared to currently available FSH products, and which preferably provides a more stable level of circulating FSH activity as compared to that obtainable by current treatment.
The present invention is directed to such products as well as the means of making such products.